Repair of large structural bone defects remains a major problem in orthopaedic reconstruction surgery due to the limited supply of cortical bone autograft and the poor long-term outcomes obtained with structural allografts. While there is no substitute for structural allografts, based on their biomechanical and biocompatible properties, their lack of osteogenic and osteoinductive potential, combined with the host's inability to remodel processed cortical bone, results in a high percentage of fractures. Towards elucidating the cellular and molecular mechanisms that regulate structural bone graft healing, we have developed a murine femoral segmental allograft model. Using this model we were able to demonstrate that autograft healing is superior to that observed with allografts due to the angiogenic and osteogenic activity from live periosteal cells. We have also developed novel approaches to engraft cells and/or recombinant adeno-associated viruses (rAAV) on the cortical surface of the femoral allografts. Based on this, we hypothesize that: 1) allograft healing and later repair is limited by the absence of angiogenic and osteogenic cells. 2) Revitalization of lifeless allograft using a cell based tissue engineering approach and/or AAV based gene therapy will confer angiogenic, osteogenic and remodeling properties on processed cortical bone leading to enhanced bone allograft healing. In our first Specific Aim, we will utilize our murine femoral bone graft model to perform transplantation of live isografts from C57B/6 Rosa 26A male mice to formally demonstrate the contributions of periosteal donor cells in structural bone graft revascularization, osteogenesis and remodeling, in our second Specific Aim, we will seed bone marrow stromal cells onto processed allograft to examine their efficacy on bone healing and remodeling of frozen allograft. In our third aim, we will combine stem cell engraftment and rAAV gene therapy to complement processed structural allograft with live cells and angiogenic-osteogenic signals, and compare its healing with autograft and allograft.